Understanding of cell death induced by the constituents of Taxus yunnanensis wood

The ethanol extract from the wood of Taxus Yunnanensis (TY) induced apoptosis in all cancer cell lines tested, which was mainly due to activation of an extrinsic pathway in human colon cancer DLD-1 cells. The extrinsic pathway was activated by the upregulation of the expression levels of Fas and TRAIL/DR5, which led to the activation of caspase-8. Of note, the machinery of this increase in expression was promoted by the upregulation of MIR32a expression, which silenced MIR34a-targeting E2F3 transcription factor. Furthermore, ectopic expression of MIR32a or siR-E2F3 silencing E2F3 increased Fas and TRAIL/DR5 expression. Thus, the extract activated the extrinsic pathway through the MIR34a/E2F3 axis, resulting in the autocrine and paracrine release of TRAIL, and upregulated expression of death receptors Fas and DR5 in the treated DLD-1 cells, which were functionally validated by Fas immunocytochemistry, and using anti-Fas and anti-TRAIL antibodies, respectively. In vivo, TY showed significant anti-tumor effects on xenografted and syngeneic model mice. The extract may also aid in chemoprevention by selectively making marked tumor cells susceptible to the tumor immunosurveillance system.

the propidium iodide (PI) staining method (Tali Cell Cycle Kit, Thermo Fisher Scientific, MA, USA). Briefly, DLD-1 cells treated with TY (5 or 10 µg/mL) or DMSO were washed twice with PBS and centrifuged at 500 × g for 5 min. These cells were fixed with ice-cold 70% ethanol. After overnight incubation at − 20 °C, the cells were centrifuged at 1000×g for 5 min at 4 °C. After a wash with 1 mL of PBS, the cells were stained with 200 μL of the Tali Cell Cycle Solution containing PI for 30 min at room temperature in the dark. Subsequently, 25 μL of stained cells was applied onto Tali Cellular Analysis Slides and analyzed with the Tali Image-Based Cytometer (Thermo Fisher Scientific), and 20 fields per sample were captured with the function of the Tali Cell Cycle Assay. The data obtained were analyzed in GraphPad Prism 8 (version 8.4.0, GraphPad Software, CA, USA).
Assay for luciferase activity. We constructed sensor vectors by joining the region with or without a possible binding site from the 3′-UTR of human E2F3 (No. 4271-4650) with a luciferase reporter pMIR-control vector (Ambion, Foster City, CA, USA) to examine the target sequence of MIR34a. To generate sensor vectors with four mutations in the binding site of the 3′-UTR of human E2F3 (No. 4440-4462) for MIR34a, we mutated seed regions from CACTGC CA to CAAGTG CA (mt-E2F3, PrimeSTAR Mutagenesis Basal Kit; TaKaRa). The sensor vector with these mutations was submitted to Life Science Research Center, Gifu University, for DNA sequencing. The cells were seeded in 12-well plates at a concentration of 0.5 × 10 5 /well the day before transfection. The sensor vector (concentration, 0.5 μg/well) and 40 nM MIR34a or nonspecific control miRNA (Dharmacon) was used for the co-transfection of the cells with Lipofectamine RNAiMAX (Invitrogen). The measurement of the activities was described previously 12 . Immunocytochemistry. After DLD-1 cells were treated with TY (4, 5, or 6 µg/mL) or DMSO for 48 h, the cells were fixed with 4% formaldehyde for 15 min and then incubated in 1% BSA/10% normal goat serum/0.3 M www.nature.com/scientificreports/ glycine in 0.1% PBS-Tween for 1 h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated overnight at 4 °C with Alexa Fluor 647 Anti-Fas antibody (ab204671, Abcam) at a 1:50 dilution. Nuclear DNA was labelled with Hoechst 33342. Images were taken by confocal microscopy (Leica-Microsystems).

Transmission electron microscopy (TEM).
After DLD-1 cells were treated with TY (5 µg/mL) or DMSO for 72 h, they were washed with ice-cold PBS. The cells were then fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.2 M PBS for 2 h. After washing with PBS and post-fixing in 2% osmium tetraoxide for 2 h, the cells were dehydrated with a 10% graded series of 30-100% ethanol and cleared with QY-1 (Nissin EM, Tokyo, Japan). The cells were then embedded in Epon 812 resin (TAAB Laboratories Equipment, Reading, UK) and sectioned at 70-nm thickness. The sections were stained with uranyl acetate and lead citrate. Transmission electron microscopic images were obtained with a Hitachi-7650 operating at 80 kV (Hitachi, Tokyo, Japan).

Animal experiments.
All mouse experiments were approved by the Institutional Animal Care and Use Committee of Gifu University and performed according to the NIH Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011). Our in vivo study was reported accordance with ARRIVE guidelines (https:// arriv eguid elines. org). Their care was in accordance with guidelines of Gifu University. All mice were maintained with ad libitum food and water under the standard light-dark cycle. Tumor volume was measured using the following formula: V = π/6(L × W2), where L, length; W, width of the tumor.
For the heterotopic colon cancer xenograft mice model, DLD-1 cells (5 × 10 6 cells/100 μL of PBS) were subcutaneously injected into the right flank of BALB/c nu/nu mice (female, 4 weeks old, SLC; n = 5). After the mean tumor volume reached approximately 100 mm 3 , the mice were randomly separated into 2 groups. Subsequently, the mice were intraperitoneally administered (once in 2 days for a total of 3 injections) TY (50 mg/ kg) or vehicle (0.1% DMSO in PBS). Two days after the final administration, the mice were sacrificed for the assessment of tumor tissues. For the heterotopic syngeneic colon cancer mice model, colon 26 cells (2 × 10 6 cells in 100 μL of PBS) were subcutaneously inoculated into female BALB/c mice (6 weeks old, SLC, Japan). After the tumor volumes reached an average of 80 mm 3 in size, the mice were randomly allocated to different groups based on their tumor size. The mice were treated with TY at 50 mg/kg or DMSO following an intraperitoneal daily administration protocol for 20 days. The mice were euthanized with an overdose of isoflurane (20 mL in an animal chamber; Pfizer, NY, USA). GraphPad Prism 8 (version 8.4.0, GraphPad Software, CA, USA) and JMP (version 12.2, SAS Institute, NC, USA) were used for statistical analysis. TUNEL staining. TUNEL staining was performed with a TUNEL assay kit (ab206386, abcam). Briefly, tissues for the TUNEL stain were fixed in 4% neutral buffered paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. The slides were deparaffinized in lemosol and rehydrated by passage through graded alcohols. For antigen retrieval, the sections were incubated with Proteinase K solution. The sections were then incubated in 0.3% hydrogen peroxide in methanol for 20 min to inhibit endogenous peroxidase activity. Subsequently, the samples were labeled with TdT enzyme and FITC. After 3 washes with TBS-T, the sections were blocked with normal serum and incubated with anti-FITC antibody conjugated with HRP. After 3 washes with TBS-T, the signals were visualized with DAB chromogen solution. The slides were counterstained with hematoxylin. Histopathological images were acquired using a microscope (BZ-X700, KEYENCE).
Statistics. Differences were statistically evaluated by one-way analysis of variance followed by the t test. A P-value of less than 0.05 was considered significant.

Results
TY induced cell death. In order to examine the growth inhibitory effects of the ethanol extract of the wood of Taxus Yunnanensis (TY), we tested 9 human cancer cell lines which have mutated p53 and 4 normal cell lines, as shown in Table 1. The activity was markedly high against the tumor cells; IC50 values ranged from 0.4 to 4 μg/mL, in contrast to the normal cells other than human fibroblast TIG-3-20 cells (Table 1). We also tested 5-fluorouracil (5-FU) and oxaliplatin (OXA)-resistant DLD-1 cells 13 (Table 1). TY exerted high growth inhibitory activity against the 5-FU or OXA-resistant DLD-1 cells, similar to the original DLD-1 cells (Table 1). Morphologically, all cancer cell lines had apoptotic findings, such as chromatin condensation and nuclear fragmentation, on Hoechst 33342 staining. In order to examine the mechanism of apoptosis, we focused on a human colon cancer DLD-1 cell line among the cell lines because it exhibited typical apoptotic changes by Hoechst 33342 staining (Fig. 1A). Cell-cycle analysis revealed a major population of sub-G1 phase after treatment with 5 μg/mL for 48 h (Fig. 1B), which indicated a typical apoptotic feature. Biochemically, the apoptosis was induced in a caspase-dependent manner, which was evaluated by Western blot analysis (Fig. 1C). The cleaved forms of poly (ADP-ribose) polymerase (PARP) that catalyzed by caspase-3 clearly emerged at 24 h after the treatment, along with the cleaved forms of caspase-8 and caspase-9, suggesting that TY induced apoptosis via both extrinsic and intrinsic pathways. Survival signal pathways of PI3K/Akt and MAPK/Erk were relatively activated up to 48 h after the treatment, but returned to normal at 72 h. The expression levels of KRAS and IκB were also unchanged during 72 h after treatment. A transition from LC3B-1 to LC3B-II, a biochemical marker of autophagy, was observed (Fig. 1C). Electron microscopic study demonstrated typical apoptotic findings accompanied by the formation of several autophagosomes in the same cell (Fig. 1D). Thus, TY induced apoptosis by activating both apoptosis pathways and altering autophagic cell death in DLD-1 cells. Next, we performed cell biological examination using caspase inhibitors. As expected, significant apoptosis inhibitory effects of Z-VAD-FMK, a pan- www.nature.com/scientificreports/ www.nature.com/scientificreports/ caspase inhibitor, were noted based on the viable cell rate and Hoechst 33342 nuclear staining ( Fig. 2A), similar to the case of the capase-8 inhibitor, Z-IETD-FMK (Fig. 2B). Furthermore, we examined the inhibitory effect of caspase-9 inhibitor, Z-LEHD-FMK on TY-induced apoptosis to verify the signaling via mitochondria. As shown in Fig. 2C, a significant inhibition of the apoptosis was found. Based on the experimental results regarding apoptosis, we next explored which death receptor(s) functioned in the extrinsic apoptosis signaling induced by the wood extract of Taxus Yunnanensis. We examined the expression of Fas/FasL and TRAIL/DR5 after treatment. As shown in Fig. 3A, the expression levels of FAS mRNA and protein in the treated cells significantly increased at 48 h and the increased protein expression level remained up to 72 h, which may lead to the upregulation of p-FADD (Fig. 3A). Thus, it is confirmed that activation of caspase-8 from death-receptors play a pivotal role in this apoptosis. Of note, the expression levels of TRAIL and DR5 markedly increased at the same time. To examine which transcription factor regulates the expression of FAS, TRAIL, and DR5, we surveyed the changes in the expression levels of certain transcription factors and tumor suppressor miRNAs by Western blot and qRT-PCR, respectively. As a result, we found markedly increased expression levels of MIR34a at 48 h after TY treatment compared with the control by qRT-PCR (Fig. 3B). Accordingly, to assess the role of MIR34a in the growth suppression along with apoptosis, we induced its ectopic expression in DLD-1 cells, which resulted in significant growth inhibition with apoptotic characteristics, such as chromatin condensation and fragmentation, on Hoechst 33342 staining (Fig. 3C). The miRNA database (miRbase) suggested that MIR34a, one of the major tumor suppressor miRNAs, silences E2F3, SIRT1, and CyclinD1. We thus examined the protein expression of the transcription factor E2F3, SIRT1, and CyclinD1 after treatment. The expression of E2F3, SIRT1, and CyclinD1 was significantly downregulated from 24 h up to 48 h (Fig. 3D). Next, the silencing of E2F3 by MIR32a (20 nM) transfection with the activation of caspase-8 was partly canceled by co-transfection with anti-MIR32a in a dose-dependent manner (Fig. 3E). Thus, the MIR34a/ E2F3 axis was established in apoptosis after the treatment with TY. Furthermore, we confirmed the increased expression levels of the death receptors DR5 and TRAIL in the MIR34a-trasfected cells at 72 h (Fig. 3F). We thus examined whether TRAIL/DR5 are the target genes of E2F3. To assess transcriptional control of DR5 and TRAIL by E2F3, we silenced E2F3 by siR-E2F3. As expected, DR5 and TRAIL were both upregulated according to Western blot analysis (Fig. 3F). Therefore, E2F3 may control the inhibitory machinery of the expression of Fas and TRAIL/DR5. The luciferase reporter assay for targeting E2F3 by MIR34a was performed and the activity was reduced by the ectopic expression of MIR34a, but partly canceled in the case of a mutated binding site ( Fig. 4A-C). As for FAS, a dose-dependent increase in the expression level was observed in the cytoplasm by immunocytochemical study (Fig. 5A).

Functional assays of the increased FAS and TRAIL/DR5 induced by TY using CH-11 and 2E5
. We next examined whether the increased expression levels of FAS and TRAIL/DR5 by TY treatment contribute to this apoptosis. Expectedly, the activating antibody for FAS (CH-11) 15,16 synergistically induced apoptosis based on the viable cell rate and Hoechst 33346 nuclear staining after combined treatment with TY (Fig. 5B). To further confirm whether TY induces TRAIL/DR5-driven apoptosis in the treated DLD-1 cells, we examined whether anti-TRAIL antibody (2E5) 11 can block the apoptosis elicited by TY. As shown in Fig. 5C, the antibody efficiently blocked the activation of caspase-8 through TRAIL/DR5 binding, similar to the case of the caspase-8 inhibitor. Thus, the upregulated Fas, TRAIL, and DR5 via MIR34a/E2F3 axis function in TY-induced apoptosis (Fig. 6).
Anti-tumor effects of TY on mouse models. In order to examine the anti-tumor effects of TY in in vivo mouse models, we produced 2 different mouse models, DLD-1 cell xenograft and syngenic tumor-bearing mice. In DLD-1 cell xenograft mice, the treatment was performed twice a week, for 4 times in total. Significant antitumor effects were found in the treated group compared with the control group (Fig. 7A). However, no apoptosis  www.nature.com/scientificreports/ in the syngenic graft tumors of treated mice was observed. In syngenic graft mice, Colon 26 cells were cutaneously inoculated into the back and the extract was intraperitoneally administered every day at a dose of 50 mg/ kg. As shown in Fig. 7B, the tumor growth in the treated group was suppressed compared with that in the control (DMSO) group. Protein samples from TY-treated tumors demonstrated the activation of caspase-3 (Fig. 7C). Pathologically, HE staining of the tumor samples confirmed the coexistence of macrophages and apoptotic cells with pyknotic nuclei in the interstitial region. TUNEL study revealed many positive cells in the area (Fig. 7D).

Discussion
We demonstrated that the natural components included in the wood of Taxus Yunnanensis induce apoptotic cell death partly through activating an extrinsic pathway, in which death ligands and receptors were upregulated by TY. TY induced apoptosis in tumor cells in an autocrine or paracrine manner via TRAIL or FasL from treated tumor cells. This suggests that the increased expression levels of Fas and TRAIL/DR5 by TY make tumor cells susceptible to attack by tumor-related immune cells such as cytotoxic T cells and NK cells. We also clarified the mechanism of upregulation of cell death ligands/receptors through the MIR34a/E2F3 axis toward apoptosis. TY markedly increased the expression level of MIR34a, which silenced E2F3 by 36-48 h after treatment. As expected, decreased expression of E2F3 by siR-E2F3 upregulated Fas and TRAIL/DR5. Thus, E2F3 negatively regulated their mRNA transcription. Furthermore, we noted increased expression of Fas in the cell cytoplasm by immunocytochemistry 48 h after the treatment with TY (Fig. 5A), and anti-Fas activating antibody (CH-11) synergistically promoted the growth inhibitory activity when combined with TY (Fig. 5B). On the other hand, the antibody against TRAIL efficiently inhibited the apoptosis elicited by TY, which suggested the release of TRAIL from the DLD-1 cells treated with TY (Fig. 5C). The MIR34a/E2F3/death ligand-receptor cascade induced by TY functions in the apoptosis. Thus, TY induced apoptosis through MIR34a/E2F3, leading to death ligand-receptor cascades. miRNAs are a group of small non-coding RNAs functioning in target gene regulation at the transcriptional or post-transcriptional level. Accordingly, miRNA can function as an oncogene or tumor suppressor by regulating growth or death-associated genes in human cancer cells. MIR34a is a typical tumor suppressor miRNA, whose transcription factor is p53, which is deleted in most cancers and DLD-1 cells 14 . The current study suggested that these bi-directional circuits exist widely among miRNA/target transcription factor genes [15][16][17] . Reinforcing the inhibition of transcripts in specific target cells is one prominent biological function of miRNAs. Some miRNAs silence E2F3 and several of them are also regulated by E2F3 through an auto-regulatory feedback loop 18 . We revealed that MIR34a targets E2F3 by Western blot analysis and Luciferase assay. As the ectopic expression of MIR34a and siRNA silencing of E2F3 both upregulated Fas and TRAIL/DR5 protein expression, miR34a/ E2F3/Fas and TRAIL/DR5 cascades may exist. Phytochemicals impact epigenetic modifications, such as DNA methylation, and histone modifications, in addition to the regulation of non-coding miRNA expression for the prevention of cancer 7,8 . Due to their significant roles in cell physiology, expression level alterations are directly related to cancer progression. In this study, the phytochemicals of TY increased the expression level of MIR34a, which may be associated with demethylation of the promoter region of MIR34a because the promoter of MIR34a is frequently hypermethylated in colon cancer cells [19][20][21] . Many phytochemicals have promise in altering DNA www.nature.com/scientificreports/ methylation and histone modifications in carcinogenesis, which may lead to the use of dietary-based phytochemicals as potent and effective chemopreventive medicines. TY induced the autocrine and/or paracrine release of FasL/Fas and TRAIL/DR5 in treated DLD-1 cells. As for TRAIL signaling, TRAIL-R2/DR5 was used for apoptosis in this case because the TRAIL antibody blocked the activation of caspase-8. The induction of apoptosis can be classified into either intrinsic apoptosis, triggered by p53 in response to cellular damages 22 , or extrinsic apoptosis induced upon death ligand binding to a death receptor. Conventional anticancer drugs induce intrinsic apoptosis through p53 in response to cellular damage 22,23 . However, most tumors have mutations in p53, leading to its inactivation and failure of chemotherapeutic agents. In contrast, TRAIL-induced apoptosis remains possible in many tumors despite non-functional p53. Therefore, the effects of TY on TRAIL signaling and sensitization may be useful for cancer prevention and therapy. Two extrinsic pathways from death-receptors can be distinguished based on the requirement of cross-signaling mitochondrial apoptotic machinery 24 . In type I cells, DISC activation is sufficient to effectively induce the full caspase cascade. In type II cells, DISC activation causes caspases-9 and -3 activation by signaling pathways via mitochondria. In this case, the mitochondria membrane potential is not downregulated and activation of caspase-9 is clearly observed. In addition, caspase-9 inhibitor considerably blocked the apoptosis. It is reasonable that the extrinsic pathway of type II is the major machinery in this apoptosis. When tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) was discovered in 1995, many studies were performed to demonstrate that it selectively induces apoptosis in tumor cells in vivo [25][26][27] . At present, TRAIL-related therapy against human cancers is under investigation. The use of TY with the ability to selectively kill tumor cells through the TRAIL-related signaling pathway is preferable. On the other hand, as shown in Fig. 1C, the expression levels of Akt/p-Akt, ERKs/p-ERKs and KRAS at 72 h after the TY treatment almost unchanged. Therefore, the survival signals from growth factors such as epidermal growth factor (EGF) and fibroblast growth factor (FGF) would sustain during activation of the extrinsic pathway. Thus, it seemed that TY does not affect the survival signal pathways. Thus, our study suggested that the wood extract from TY selectively kills tumor cells by inducing apoptosis through the increased expression of cell-death ligands/receptors, which also make the marked tumor cells susceptible to the immunosurveillance system. The similar findings were also observed in other colon cancer cell lines.
TY contains many diterpenoids, such as baccatin III, 10-deacetyltaxuyunnanine C, and taxuyunnanine E, which may have anti-proliferative effects 5 . However, most are cytotoxic to healthy cells. Cytotoxic 10-deacetyltaxuyunnanine C induced the activation of caspase-8, but not the increased expression of cell-death factor/receptors. The anti-cancer effects of TY may be due to synergy between diterpenoids and other factors such as lignans.